Lanthanoid complex capsule and particle contrast agents, methods of making and using thereof

ABSTRACT

The invention relates to compositions of DOTA derivative compounds, lanthanoid-DOTA derivative molecular complex, and lanthanoid-complex encapsulated solid lipid particles or capsules, and methods of making and using the compositions. The solid lipid particles or capsules contain micelle cores stabilized by a hyperbranched polymer shell based from a crosslinked DOTA derivative compound or crosslinked lanthanoid-DOTA derivative complex. These solid lipid particles or capsules can be used in various applications, such as contrast agents or drug delivery vehicles.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/482,950, filed May 5, 2011 and U.S. ProvisionalPatent Application Ser. No. 61/604,770, filed Feb. 29, 2012, both ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to lanthanoid-complex encapsulated solidlipid nanoparticles, or lanthanoid-complex nanoparticles, methods ofmaking and using thereof.

BACKGROUND OF THE INVENTION

Biomedical imaging, such as magnetic resonance imaging (MRI), is acompelling imaging method as it allows peering into body withnon-ionizing radiation. The images thus acquired enable the diagnosisand treatment of conditions that were previously revealed only by thescalpel. Generating new nanoscale imaging agents that can provideprecise and high contrast views of lesions that would guide focused andeffective treatment is certainly going to benefit biomedical imaging.

Multimodal nanoparticles are great vehicles with which to achievesimultaneous targeting, imaging, and drug delivery, an important goal inmodern pharmacology (BHANU P. S. CHAUHAN: HYBRID NANOMATERIALS:SYNTHESIS, CHARACTERIZATION, AND APPLICATIONS, (John Wiley & Sons,Hoboken, N.J., 2011)). As part of this effort, an MRI imaging agentcomprised of gadolinium complexes has recently been assembled on thesurface of silver nanoparticles (Siddiqui et al., J. Colloid Interf.Sci. 337:88 (2009)). Noble metal NPs have been used in the developmentof multifunctional agents for the diagnosis and/or treatment of disease,because noble nanoparticles such as gold and silver nanoparticles areeasy to synthesize. Such particles can be made multifunctional bysupporting moieties anchored on their surface.

However, certain drawbacks exist in the use of noble metal particles forroutine clinical use. For instance, these particles are expensive, andthe use of these particles is inefficient because of an unused interiorvolume. Moreover, metal particles above 5-8 nm diameters are clearedonly slowly through a hepatic pathway.

Solid lipid nanoparticles (SLNs) offer an important alternative for theformation of multimodal theranostic agents (Lammers et al., Acc. Chem.Res. 44(10):1029 (2011); Andreozzi et al., Bioconjugate Chem. 22:808(2011)). They have certain characteristics that suit them for combineddrug delivery and diagnostics (Muller et al., Eur. J. Pharm. Biopharm.50:161 (2000)). Gd-containing SLNs has been reported as MRI contrastagents (Morel et al., Eur. J. Pharm. Biopharm. 45:157 (1998)). In thatreport, the uptake of gadoliniumdiethylenetriamine-N,N,N′,N″N″-pentaacetate (GdDTPA) and gadoliniumtetraazacyclododecanetetraacetic acid (GdDOTA) to form the contrastagents was described, but the question of the contrast agent's locationin relation to the surface of the SLN particle was left open. As aresult, the mechanisms of contrast enhancement by Gd-containing SLNscould not be specified. In this type of particle, the subsurface/surfacelocation of the contrast agent determines the degree to which protonspin relaxation of water is due to inner sphere, T₁ relaxation (surface)or outer sphere T₁ and T₂, or susceptibility relaxation (subsurface)(Fossheim et al., J. Magn. Reson. Imaging 7:251 (1997)). In a follow-upstudy, a lipid with a polar GdDTPA headgroup was embedded in an SLNsurface with GdDTPA confined to the surface (Zhu et al., J. Nanosci.Nanotechnol. 6:996 (2006)). A more recent paper describes theincorporation of the [GdDTPA]²⁻ complex within an SLN core for magneticresonance colonography. The latter particles were prepared in aminiemulsion in which [GdDTPA]²⁻ was introduced in an aqueous phase thatalso contained monostearin (Sun et al., Magn. Reson. Med. 65:673(2011)). It has also been described that the incorporation of neutralgadolinium acetylacetonate (GdAcAc) in SLNs, generated by nanotemplateengineering, can be used for crossing the blood brain barrier in neutroncapture therapy of brain lesions (Oyewumi & Mumper, Bioconjugate Chem.13:1328 (2002)).

The SLN platform has been found advantageous in the above studies from aclinical perspective because of its nanoscale size, biocompatibility,and biodegradation properties that aid clearance. However, theapplication of SLNs as imaging agents, possibly combined with drugrelease as theranostic agents, still has not been well developed.

In the United States, MRI contrast agents employ the DTPA ligand and itsderivatives to coordinate the contrast-inducing ion Gd³⁺. DTPA isconstructed from a diethylene triamine backbone. One attractive featureof DTPA is the ease with which it can be chemically modified to adjustits pharmacokinetics and biodistribution. However, GdDTPA with astability constant of 10²² M⁻¹ has a significant toxicological drawback.It has been shown to induce nephrogentic systemic fibrosis (NSF) acondition that is sometimes fatal resulting from the release of Gd³⁺ions due to renal insufficiency (see Bongartz, Magn. Reson. Mater. Phy.20:57 (2007), which is hereby incorporated by reference in itsentirety). GdDOTA is more stable by several orders of magnitude with astability of constant of 10²⁸ M⁻¹ (see Magerstadt et al., Magnet. Reson.Med. 3:808 (1986), which is hereby incorporated by reference in itsentirety). What is therefore needed is a GdDOTA-based agent thatcontains functional groups based on DOTA ligands that enables thecompositions to be useful in MRI applications. This invention answersthat need.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a compound of formula (I)or (II):

In formula (I) or (II), R is H, alkyl, or a cation; R′ is H, or apolymer precursor group which is able to self-crosslink or crosslink inpresence of one or more crosslinking agents.

Another aspect of the present invention relates to a molecular complexcomprising a molecule of formula (III):

and optionally a balancing counterion. In formula (III), M is a chelatedmetal ion and R′ is H, or a polymer precursor group that is able toself-crosslink or crosslink in presence of one or more crosslinkingagents.

Another aspect of the present invention relates to a hyperbranchedpolymer nanoparticle or nanocapsule comprising hyperbranched polymericunit having formula (IV):

and optionally a crosslinking moiety CL. In formula (IV), R is H, alkyl,or a cation; X is O or S; n is 1-10; p is the number of the units offormula (IV) ranging from 2 to 200,000; and crosslinking moiety CL, ifpresent, is a moiety connecting the hyperbranched polymeric unitstogether through covalently bonding to each X of formula (IV).

Another aspect of the present invention relates to a hyperbranchedpolymer nanoparticle or nanocapsule comprising a hyperbranched polymericunits having formula (V):

and optionally a crosslinking moiety CL. In formula (V), M is a chelatedmetal ion; X is O or S; n is 1-10; p is the number of the units offormula (V) ranging from 2 to 200,000; and crosslinking moiety CL, ifpresent, is a moiety connecting the hyperbranched polymeric unitstogether through covalently bonding to each X of formula (V).

A series of DOTA derivative contrast agents have been developed toprovide a stable contrast agent while incorporating additional groupsthat enable modification for enhanced (1) bioavailability, (2)pharmacokinetics, and (3) specificity. In particular, a class ofnanoscale MRI contrast agent has been synthesized to provide highcontrast of MRI images by assembling contrast-inducing ions in a clusterof sites on hyperbranched polymeric particles. The method employs chargepairing between cationic surfactants and anionic monomers that yieldcompact polymeric structures with or without the aid of coupling agents.The hyperbranched polymeric particles are formed from metal complexescontaining a chelated metal ion, for instance, a lanthanoid such as Gd³⁺or Eu³⁺, with DOTA or its derivative as the monomer and metalcoordination site. The cationic solid lipid nanoparticles were formedwith metal-DOTA derivatives complexes as couterions on the surface ofthe nanoparticles, the pendant hydroxyl groups on metal-DOTA derivativecomplexes act as precursors for Stern layer polymerization with orwithout the aid of crosslinking agents.

The synthetic method by which the contrast agents were produced isflexible and allows for the use of molecular groups that target specifictissues, enhance solubility, and shield the agents from the immunesystem. A large number of modifications can be made on the DOTA-basedligand system by the method described herein to prepare additional newMRI/PET agents.

The hyperbranched polymer-based particles and capsules are multimodalstructures that can carry a high concentration of contrast enhancing andimaging agents. They are organic nanostructures that are chemicallyrelated to single molecule complexes currently used for biomedicalimaging. The compact polymeric nanoparticles are robust and are suitablefor biomedical imaging, catalysis, and materials synthesis.

The hyperbranched polymer based particles also allow the incorporationof drugs within the particle core or appending drug molecules to theparticle surface: the particles are approximately 5-10 nm in size, smallenough for direct clearance through the kidneys, thus are suitable as adrug delivery vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts exemplary crosslinkers and corresponding crosslinkingreactions for an exemplary compound DhS-OH. DhS refers to DOTAhomoserine.

FIG. 2 is a scheme depicting an exemplary process for forming ahyperbraned polymer nanoparticle or nanocapsule.

FIG. 3A is a simplified scheme depicting a mixed micelle stabilized bycombining the cationic surfactant cetyltrimethylammonium chloride (CTAC)with the neutral surfactant polyethylene glycol lauryl ether (Brij 35)on its surface. FIG. 3B depicts the detail of hyperbranched polymericcapsule assembly (solid lipid nanoparticle) comprising a mixed micellecore surrounded by a hyperbranched polymeric shell after crosslinking[LnDhS]⁻ of the cationic surfactant with a crosslinking agent, DVS.

FIG. 4 is a schematic illustration of the process of CTA[EuDhS]_(CL)SLNsynthesis where CL refers to a crosslinking moiety and EW denotes theemulsifying wax core.

FIG. 5 is a graph showing the fluorescence spectra of NaCl(aq)-treated[EuDhS]SLN (red), and the salt-free control [EuDhS]SLN (green).

FIG. 6 is a graph showing the fluorescence spectra of NaCl(aq)-treated[EuDhS]_(CL)SLN (red) and the salt-free control [EuDhS]_(CL)SLN (green).

FIG. 7 is a graph showing TEM image of SLN particles. (Bar represents100 nm).

FIG. 8 is a graph showing the proton NMR spectrum of CTA[EuDhS]_(CL)SLNin D₂O. The insets show the proton NMR spectrum of K[EuDhS] in D₂O.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a compound of formula (I) or(II):

Another aspect of the invention relates to a molecular complexcomprising a molecule of formula (III):

and optionally a balancing counterion.

In formula (I) or (III), R is H, alkyl, or a cation. For instance, R canbe H, Li⁺, Na⁺, K⁺, NH₄ ⁺, R″NH₃ ⁺R″₂NH₂ ⁺R″₃NH⁺, where R″ can be analkyl.

R′ is H, or a polymer precursor group which is able to self-crosslink orcrosslink in presence of one or more crosslinking agents. For instance,R′ can be H or a substituted alkyl group. Examples for R′ group includehydroxyalkyl, alkylthioalkyl, and mercaptoalkyl or a radical thereof. Inone embodiment, R′ is —CH₂—OH, —C₂H₄—OH, —C₂H₄—SH or —C₂H₄—S., or—C₂H₄—S—CH₃.

In formula (III), the metal ion M is a chelated metal ion, and can beany metal that can be chelated in the macrocycle structure of formula(III), known to one skilled in the art. For instance, the metal ion Mcan be a lanthanoid or an 111A atom. Examples for M are La, Eu, Tb, Gd,Ga, and In.

The compounds of formula (I) and (III) are considered1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or itsderivatives. DOTA or its derivatives can be prepared by reacting cyclen,having the chemical formula

(1,4,7,10-tetraazacyclododecane) with a precursor compound underconditions effective to provide functional groups as substituents on theN atoms on cyclen. For instance, bromobutyrolactone can be used as aprecursor compound that reacts with cyclen under mild conditions (i.e.,at atmospheric pressure and room temperature) to add butyrolactone oneach N atom on cyclen, resulting in cyclen tetrabutyrolactone (i.e., thecompound of formula II). Base hydrolysis of the lactone then producesDOTA homoserine (DhS) (1), as shown in formula (I) where R=—H,R′=—CH₂CH₂OH. The homoserine hydroxyl group can serve as the site forfurther derivativization. For instance, the side chains can be modifiedto produce thiol terminal groups (see Lazar et al., Eur. J. Org. Chem.351 (2002), which is hereby incorporated by reference in its entirety).

The compounds of formula (I) can be either chiral or anchiral.

As shown in Scheme 1, one DOTA derivative is available as an achiralcompound, DOTA(homoser)₄, (DhS) (1) which is derived from cyclentetrabutyrolactone (see Lazar et al., Eur. J. Org. Chem. 351 (2002),which is hereby incorporated by reference in its entirety).

Two additional compounds DOTA(ser)₄, (DS) (2) and DOTA(met)₄, (DM) (3)are modifications of DOTA in which the side chains of serine andmethionine, respectively, extend from the a carbon of the four acidgroups of DOTA (Scheme 1). The products are formed from reactionsbetween cyclen and 2-bromo-3-hydroxypropanoic acid (from serine) and2-bromo-4-(methylthio)butanoic acid (from methionine), respectively.Both DS and DM are chiral and can be modified at the acidic side chainsto modulate their bioavailability, or they can be incorporated intolarger structures such as oligomers, polymers, and proteins.

The compounds of formula (I) can be used as complexing agents to chelateions. In particular, the compound of formula (III) is a molecularcomplex formed from DOTA derivatives chelating a metal ion M, such aslanthanoids and other metal ions. Such molecular complexes can be usedas for biomedical imaging, catalysis, sensors, and materials synthesis.For example, compounds 1 and 2 can be assembled to form nanoparticlesthrough ether or ester bond formation between adjacent DOTA complexes bydivinyl sulfone (DVS) or dicarboxylic acids, respectively. Compound 3can be demethylated at sulfur to form a precursor complex that can beoligomerized through the formation of disulfide bonds. This precursorcompound can be employed as a building block formetal-organic-frameworks (MOFs) wherein the thioethers can bond tosuitable metals such as Ag⁺, Cu⁺ or Zn²⁺.

The chelation structures of formula (III) form eight coordinatetetramino-tetracarboxylato metal complexes. In each resulting complex,four a carbon pendant groups remain available for further derivatizationor crosslinking reactions. For instance, the ligands DS, DM, and DhS canincorporate Ln³⁺ ions to produce molecular complexes, e.g.,[LnDOTA(ser)₄]⁻.

The ligand DhS forms analogous complexes with hydroxy ethyl groups atthe a carbon of each of the four carboxylic acids of the ligand. Theseligands are precursors for more elaborate molecules with attributes thatinclude high specificity, biocompatibility, desirable pharmacokineticproperties and the like. They are also monomer precursors tohyperbranched polymeric nanoparticles and nanocapsules. The hydroxygroups of DS or DhS can be linked to the same or different adjacentmonomers.

The structures of compounds of formula (I) and (II) or molecularcomplexes of formula (III) obtainable by the above routes can formhyperbranched polymeric capsules, or particles. These particles orcapsules can be prepared from homogeneous monomers or prepared by mixingdifferent monomers. They can be used homogeneously or mixed withdifferent components.

An additional aspect of the present invention provides a method ofmaking a hyperbranched polymeric nanoparticle or nanocapsule. The methodcomprises connecting the compounds of formula (I) or molecular complexesof formula (III) described above through modifying the functional groupof R′. The modifications include crosslinking the compounds or molecularcomplexes described above with their neighboring compounds or molecularcomplexes through bond formations, such as ether, ester or disulfidebond formations.

In one embodiment, the R′ group is hydroxyalkyl. The compound of formula(I) and (II) or molecular complex of formula (III) is bonded to theadjacent compounds or molecular complexes through one or morecrosslinking agents.

In one embodiment, the R′ group is mercaptoalkyl or radical ofmercaptoalkyl. The compound of formula (I) and (II) or molecular complexof formula (III) is bonded to adjacent compounds or molecular complexesthrough disulfide bond formations. For instance, the R′ group can be analkylthioalkyl, and the connecting step comprises removing the terminalalkyl group of R′, and the remaining terminal sulfide is used to formdisulfide bonds between compounds or molecular complexes.

Accordingly, one aspect of the invention relates to a hyperbranchedpolymer nanoparticle or nanocapsule comprising: hyperbranched polymericunits having formula (IV):

andoptionally a crosslinking moiety CL.

Another aspect of the invention relates to a hyperbranched polymernanoparticle or nanocapsule comprising: hyperbranched polymeric unitshaving formula (V):

and optionally a crosslinking moiety CL,

In formula (IV) or (V), R is H, alkyl, or a cation. For instance, R canbe H, Li⁺, Na⁺, K⁺, NH₄ ⁺, R″NH₃ ⁺R″₂NH₂ ⁺R″₃NH⁺, where R″ can be analkyl. X is O or S. The variable n is 1-10. For instance, n is 1-6, 1-4or 1-2. The variable p is the number of the units of formula (IV) or (V)ranging from 2 to 200,000. In one embodiment, p ranges from 10 to 5000,for instance from 20 to 400, for hyperbranched polymer nanoparticleformed from the hyperbranched polymeric units (IV) or (V). In anotherembodiment, p ranges from 10,000 to 200,000, for instance from 18,000 to170,000, for hyperbranched polymer nanocapsule formed from thehyperbranched polymeric units (IV) or (V).

The hyperbranched structures form by crosslinking. The crosslinkingmethod is similar to crosslinking copolymer blocks to form micelles. Seefor example, Read & Armes, Chem. Comm. 3021 (2007), which is herebyincorporated by reference in its entirety.

The hyperbranched polymeric units having formula (IV) or (V) are formedby crosslinking the monomeric form, which is compound of formula (I), or(III), in the presence or absence of one or more crosslinking agent.Crosslinking reaction can happen directly by linking the R′ groups ofthe formula with the adjacent compounds or molecular complexes, asdescribed above. Alternatively, the R′ groups of the formula can bemodified to present functional group that is capable of crosslinkingreaction to bond with the R′ groups or modified R′ groups of theadjacent compounds or molecular complexes.

Methods of modifying R′ groups and crosslinking reactions between the R′groups or modified R′ groups to form a hyperbranched polymernanoparticle or nanocapsule are those known to one skilled in the art.For instance, crosslinking reaction can occur through ether or ester,where R′ or modified R′ has a terminal —OH group, or disulfide bondformations where R′ or modified R′ has a terminal —SH or —S. group.

Suitable crosslinking agents include, but are not limited to, divinylsulfone (DVS), dicarboxylic acid, diepoxybutane, diepoxyoctane,divinyladipate (DVA), epichlorohydrin, butanediol-diglycidyl ether(BDDE), ethylene glycol diglycidyl ether, polyglycerol polyglycidylether, ethylene sulfide, glutaraldehyde, bromoacetic anhydride, acrylicanhydride, 3-mercaptopropanoate, thioacetic acid, or combinationsthereof. Additional descriptions for modifications of R′ group andsuitable crosslinking reactions/crosslinking agents can be found inSchanté et al., Carbohydate Polymers 85:469-89 (2011), which is herebyincorporated by reference in its entirety.

The crosslinking moiety CL of formula (IV) or (V), if present, is amoiety connecting the hyperbranched polymeric units together throughcovalently bonding to each X of formula (IV) or (V). Exemplarycrosslinking reactions for an exemplary compound DhS-OH includingvarious CLs to form hyperbranched polymeric units are shown in FIG. 1.An exemplary scheme of forming a hyperbraned polymer nanoparticle ornanocapsule is shown in FIG. 2.

One or more catalyst can be used in the crosslinking reaction. Forinstance, a disulfide forming catalyst, FeNTA, can be used for disulfideformation (Walters et al., Inorg. Chim. Acta 359:3996 (2006), which ishereby incorporated by reference in its entirety).

The crosslinking reactions of compounds or molecular complexes offormula (I), (II) or (III) form the hyperbraned polymer shell containingthe hyperbranched polymeric unit having the formula (IV) or (V).

In one embodiment, all the terminal groups of all compounds or allmolecular complexes of formula (I), (II) or (III), i.e., all four R′groups or modified R′ groups, have been crosslinked with R′ groups ormodified R′ groups of neighboring compounds or molecular complexes.Crosslinking compounds or molecular complexes of formula (I), (II) or(III) therefore form a completely enclosed hyperbraned polymer shellshowing by the hyperbranched polymeric units of formula (IV) or (V).

In another embodiment, not all terminal groups of all compounds or allmolecular complexes of formula (I), (II) or (III) have been crosslinkedwith the neighboring compounds or molecular complexes. In this regard,the hyperbraned polymer nanoparticle or nanocapsule then contain notonly the hyperbranched polymeric units of formula (IV) or (V), but alsothe terminal compounds or terminal molecular complexes which haveterminal functional groups that have not been crosslinked with theneighboring compounds or molecular complexes. These terminal compoundsor terminal molecular complexes then can have formula (I), or (III),with one, two or three R′ groups crosslinked with the hyperbranchedpolymeric units of formula (IV) or (V). For instance, in FIG. 2, thehyperbraned polymer capsule 1 contains a hyperbranched polymeric unitthat can be represented by formula (V) in the central, and four terminalmolecular complexes having formula (III), each of which has one R′ groupcrosslinked with the central hyperbranched polymeric unit and threeterminal R′ groups that have not been crosslinked with other molecularcomplexe.

In one embodiment, X is S; formula (IV) or (V) is connected to eachother through disulfide bond without the aid of crosslinking agent. Thusformula (IV) or (V) does not contain the crosslinking moiety CL.

In one embodiment, X is O; formula (IV) or (V) is connected to eachother with the aid of one or more crosslinking agents.

The metal ion M is in formula (V) is the same as it is defined informula (III), and has been described above.

For example, lanthanoid complexes of the DOTA derivative DM have fourmethyl sulfido ethyl groups at the a carbon of the ligand acids. Themethyl groups can be removed in sodium/liquid ammonia solution to formDOTA(homocysteine)₄, (DhC). Lanthanoid complexes of this ligand,LnDOTA(homocys)₄ can be converted to a hyperbranched polymer shellthrough disulfide bond formation.

Some embodiments of the present invention also provide a hyperbranchedpolymer shell comprising the crosslinked hyperbranched polymericnanoparticles or nanocapsules described in the above embodiments.

Another aspect of the present invention relates to a mixed micelle or asolid lipid nanoparticle formed from the mixed micelle. The mixedmicelle comprises micelle core, formed by such as an emulsifying wax;and at least one cationic surfactant associated to the surface of themicelle core.

Any surfactants can be used to form the micelle core. Exemplary micellecore is formed from an emulsifying wax; a lipid, such as tricaprin,trimyristin, tripalmitin, tristearin; hydrogenated coco-glycerides; or ahard fat, such as WITEPSOL®, MASSA ESTARINUM®, or Compritol® 888 ATO; astearic acid; or a soybean lecithin.

Any cationic surfactant known to one skilled in the art for formingsolid lipid nanoparticle or nanocapsule can be used to prepare thecationic surfactant associated to the surface of the micelle core.Suitable cations of the cationic surfactant include, but are not limitedto, alkyltrimethylammonium such as cetyltrimethylammonium;dimethyldioctadecylammonium; dioctadecyldimethylammonium, etc. Suitablesources of these cations of the cationic surfactant include, but are notlimited to, alkyltrimethylammonium salts: such as cetyltrimethylammonium bromide (CTAB) or cetyl trimethylammonium chloride(CTAC); cetylpyridinium chloride (CPC); dimethyldioctadecylammoniumchloride; dioctadecyldimethylammonium bromide (DODAB);cetyldimethylammonium acetamide bromide; or other cationic surfactantalike, including lipids.

The anionic portion of the cationic surfactant is the hyperbranchedpolymeric nanoparticle or nanocapsules described in the aboveembodiments.

The mixed micelle may further comprise at least one neutral surfactant.The molecular chains of this neutral surfactant can intersperse theindividual molecules of cationic surfactant. For instance, the neutralsurfactant can be a polyethelene glycol lauryl ether. In one embodiment,the neutral surfactant is Brij L23, which is a PEG-containing diblockcopolymer surfactant.

The SLNs formed herein are enclosed, or at least partially enclosed, bythe hyperbranched shell. In an exemplary embodiment, the particle orcapsule described herein imbed cationic surfactants such as CTAB orCTAC, and neutral surfactants such as Brij surfactant in the SLN surfaceand then exchange Br of CTAB or Cl of CTAC with [MDOTA]⁻. Theintroduction of DVS as a linker monomer leads to the formation of ahyperbranched—{[LnDOTA]⁻-DVS}_(n)—shell above the SLN and nestled belowthe polyether arms of Brij L23 that extend outward from the surface ofthe SLN. See FIGS. 3A-3B. The polyether arms confer water solubility onthe particle and serve as a steric barrier to inter-particlecrosslinking.

The hyperbranched shell encapsulating SLNs can have a dispersed sizerange, from 5 nm to 500 nm. In one embodiment, the average size of thenanoparticle or nanocapsule ranges from about 50 to about 150 nm.

Another aspect of the present invention relates to a method of preparinga mixed micelle or a solid lipid nanoparticle formed from a mixedmicelle. The method comprises formulating a micelle core by mixing anemulsifying wax, at least one neutral surfactant, and at least onecationic surfactant, wherein the anionic portion of the cationicsurfactant is the molecular complex of formula (I) or (III);crosslinking the molecular complexes by modifying the functional groupof R′, optionally bonded through one or more crosslinking agents, toform a hyperbranched polymeric shell around the micelle core, therebyforming a solid lipid nanoparticle. The hyperbraned polymeric shellaround the micelle core contains the hyperbranched polymeric unit havingthe formula (IV) or (V), and has been described above.

When large particles or aggregates are present when formulating themixed micelle, the reaction mixture can be filtered before thecrosslinking step to remove these large particles or aggregates.

The micelle core, neutral surfactant, and anionic portion and cationicportion of the cationic surfactant have been described in theembodiments above. In one embodiment, the cationic portion of thecationic surfactant is cetyltrimethylammonium, the anionic portion ofthe cationic surfactant is a hyperbranched polymer shell formed frommolecular complex of formula (I) or (III), wherein the metal is alanthanoid, and the neutral surfactant is polyethelene glycol laurylether.

In an exemplary embodiment, when base hydrolysis was carried out withcetyltrimethylammonium hydroxide (CTA⁺OH⁻), the addition of LnCl₃results in a monoanionic lanthanide complex [LnDhS]⁻ with CTA as itscounterion (Scheme 2) as a precursor for capsule assembly.

The mixed micelles can be stabilized by combining the cationicsurfactant cetyltrimethylammonium bromide (CTAB) with the neutralsurfactant polyethylene glycol (23) lauryl ether (Brij 35) (FIG. 3A).The replacement of bromide anion with two or more reactive functionalgroups allows for the preparation of anionic polymers below the coronal(terminal block) region of mixed micelles. An additional description forforming mixed micelle from surfactant can be found in Gao et al., J.Colloid Interf. Sci. 273:626 (2004), which is hereby incorporated byreference in its entirety.

The substitution of DS or DhS (monomer A) for the Br ion of CTAB or theCl⁻ of CTAC in the Gao-type mixed micelles, and the introduction of DVS(monomer B) as a crosslinker allows the formation of hyperbranched ABpolymer networks. In the initial mixed micelle, there is a shell ofanions that are closely spaced within the micelle core and thereforedistal to adjacent micelles, as enforced by the polyethyl ether chain ofBrij 35. The neutral crosslinker induces polymerization in the annularregion of the micelle where the anionic groups are paired with thecationic headgroups of CTA (FIG. 3B).

The formation of core-crosslinked micelles can be prepared with di- andtri-block copolymers where one of the blocks has polar hydroxyl groupsthat form a region suited to accept polar monomers and/or crosslinkers(see Read & Armes, Chem. Comm. 3021 (2007); Liu et al., Langmuir 18:7780(2002); Gao et al., Chem. Mater. 20:3063 (2008), which are herebyincorporated by reference in their entirety). In the Gao-type mixedmicelle, the polar region is the site of the surfactant headgroup. Thusthe polar region is explicitly ionic and suited for the introduction ofcharged monomers or crosslinkers. The specific assembly of anionicpolymers in mixed micelles is enabled by (1) the hydrophobicity of thehexadecyl alkane tail of the CTA surfactant, the dodecayl tail of Brij35 which form the micelle core, (2) the charge pairing of the DOTAanions with the cationic trimethyl ammonium head group of CTA, and (3)the stabilization of the CTA DOTA system by interspersed unchargedsurfactants, Brij 35, that sterically prevents the crosslinking betweenadjacent micelles. The surfactant monomer charge pairing occurs withinthe recessed regions of the micelle. In sum, the system segregates theanions in an intermediate region of the micelle by the combined effectsof CTA micelle formation, charge pairing, and the diblockcharacteristics of Brij 35 that stabilizes the hydrophobic core andsterically isolates the annular ionic region of the micelle.

Shell formation from the crosslinking of DOTA complexes generatesstabilized micelles because the DOTA is simultaneously locked in placeby charge pairing with anions in the shell and the Brij surfactant isobserved to remain in the structure, yielding a water soluble particle.After the solid lipid nanoparticles are formed and the hyperbranchedpolymeric shells are formed around the micelle core, the neutralsurfactant can be removed from the solid lipid nanoparticle by asolvent. For instance, Brij may be removed with the appropriate solvent,which leaves a hydrophobic crosslinked shell.

Crosslinking between functional groups of monomers leads to stablemicelle formation without inter-micelle crosslinking to form undesirableparticle dimers, trimers, or clusters. The process may be described asemulsion surface polymerization (ESP). Through ESP, a large variety ofanionic compounds can be employed for analogous capsule formation onSLNs, liposomes, other nanoparticles or surfaces.

This synthetic approach can be applied to the vast array of anionicmonomers and can be of great commercial importance in the assembly ofdiagnostic, theranostic, or catalytic nanoparticles. Synthesis ofpolymers within simple mixed micelles is cost effective and could havelimitless applications.

Mixed micelled components can also form a reverse micelle. In oneembodiment, the reverse micelles contain the cationic surfactant, suchas cetyldimethylammonium acetamide (CDA):

[Fe(CN)₆]³⁻ (see Walters et al., Inorg. Chem. 44:1172 (2005);Mehltretter, J. Am. Oil Chem. Soc. 44:219 (1967), both of which arehereby incorporated by reference in their entirety). In this embodiment,CDA⁺ is employed to form the salt CDA₄-[DhS](see Scheme 2 for a similarstructural representation of CDA₄-[DhS], wherein CDA⁺ replaces CTA⁺).The CDA₄-[DhS] salt dissolves in organic solvents to form amicroemulsion composed of reverse micelles with the formula unitCDA₄-[DhS], wherein the DhS anions occupy a volume that is enclosed byCDA cations. The volume occupied by DhS anions may be dry (see Walterset al., Inorg. Chem. 44:1172 (2005) or may incorporate water in themanner of classical reverse micelles. The microemulsion serves as aprecursor for the preparation of hyperbranched polymeric nanoparticlesof DhS.

Embodiments of the present invention also relate to a contrast agentcomprising the compound or molecular complex of formula (I) or (III),the hyperbranched polymeric nanoparticle or nanocapsule formed from thehyperbranched polymeric unit of formula (IV) and (V), or the mixedmicelle described herein.

Accordingly, one aspect of the invention relates to a method of usingthe compound or molecular complex of formula (I)-(III), thehyperbranched polymeric nanoparticle or nanocapsule formed from formula(IV) and (V), or the mixed micelle described herein as a contrastagents. These contrast agents can be used in various areas, includingbut are not limited to, magnetic resonance imaging (MRI) analysis,nuclear magnetic resonance (NMR) analysis, florescence analysis,positron emission tomography (PET) analysis.

Lanthanoid ions can be selected for diamagnetism (La³⁺) for NMRanalyses, fluorescence properties (Eu³⁺) or MRI contrast (Gd³⁴). Maingroup isotopes ⁶⁸Ga³⁺ or ¹¹¹In³⁺ can be incorporated in the monomers forPET.

In one embodiment, the molecular complex of formula (III), thehyperbranched polymeric nanoparticle or nanocapsule formed from formula(V), or the mixed micelle described herein can be used as a contrastagent in MRI analysis. An exemplary chelated metal used in theseformulas is Gd.

In one embodiment, the molecular complex of formula (III), thehyperbranched polymeric nanoparticle or nanocapsule formed from formula(V), or the mixed micelle described herein can be used as a contrastagent in NMR analysis. An exemplary chelated metals used in theseformulas is La.

In one embodiment, the molecular complex of formula (III), thehyperbranched polymeric nanoparticle or nanocapsule formed from formula(V), or the mixed micelle described herein can be used as a contrastagent in analyzing florescence properties. Exemplary chelated metalsused in these formulas are Eu and Tb.

In one embodiment, the molecular complex of formula (III), thehyperbranched polymeric nanoparticle or nanocapsule formed from formula(V), or the mixed micelle described herein can be used as a contrastagent in PET analysis. Exemplary chelated metals used in these formulasare Ga and In, for instance, ⁶⁸Ga and ¹¹¹In.

An embodiment of the invention relates to a method of biomedicalimaging. The method comprises administering to a patient the molecularcomplex of formula (III), the hyperbranched polymeric nanoparticle ornanocapsule formed from formula (V), or the mixed micelle describedherein; and conducting a biomedical imaging test on the patient.

Another embodiment of the invention relates to a method of delivering ahigh concentration of contrast enhancing and/or imaging agents thatcomprises the step of administering to a patient the molecular complexof formula (III), the hyperbranched polymeric nanoparticle ornanocapsule formed from formula (V), or the mixed micelle describedherein as a contrast agent. The chelated metal acts as the contrastenhancing and/or imaging agent.

Solid lipid nanoparticles (SLNs) offer an important route for theformation of multimodal theranostic agents, compared with noble metalparticles. They are natural choices for the formation of theranosticagents because of their proven capacity to deliver drugs, andincorporate diagnostic molecules either on their surface or in theirinterior. The particle has been long valued for its utility in drugdelivery particularly across the blood brain barrier (BBB). Theseparticles are 50-200 nm in diameter and are composed of biocompatiblelipids that are solid under ambient conditions. SLNs are inexpensive andoften undergo disintegration in solution at rates that can be tailoredto facilitate clearance.

Thus, embodiments of the invention relate to the hyperbranched shellencapsulating SLNs comprising the mixed micelle described above and atherapeutic agent. The therapeutic agent can be encapsulated within themixed micelle. Additionally, the hyperbranched shell encapsulating SLNsdescribed herein can be used as a drug delivery vehicle.

Accordingly, an embodiment of the invention also relates to a method ofdelivering drugs across physiological barriers, such as blood-brainbarrier. The method comprises administering to a patient thehyperbranched shell encapsulating solid lipid nanoparticle describedherein. The size of the hyperbranched shell encapsulating solid lipidnanoparticle for drug delivery typically ranges from 50-150 nm, or from50-100 nm.

Any therapeutic agent known by those of skill in the art to havetherapeutic activity can be contrained in the solid lipid nanoparticles.Suitable therapeutic agents include, but not limited to, chemicals,cells (including stem cells), proteins, peptides, nucleic acids (e.g.,DNA, RNA, siRNA), nucleic acid analogues, nucleotides, oligonucleotidesor sequences, peptide nucleic acids (PNA), aptamers, antibodies orfragments or portions thereof (e.g., paratopes orcomplementarity-determining regions), antigens or epitopes, hormones,hormone antagonists, cell attachment mediators (such as RGD), growthfactors or recombinant growth factors and fragments and variantsthereof, cytokines, enzymes, antioxidants, antibiotics or antimicrobialcompounds, anti-inflammation agents, antifungals, viruses, antivirals,toxins, prodrugs, drugs, dyes, amino acids, vitamins, chemotherapeuticagents, and small molecules. The agent may also be a combination of anyof the above-mentioned therapeutic agents.

In one embodiment, the therapeutic agent is a drug suitable for beingdelivered across physiological barriers, such as the blood-brainbarrier.

In one embodiment, the therapeutic agent is an antibiotic or anti-tumoragent. Exemplary antibiotic agents include, but are not limited to,doxorubicin; actinomycin; aminoglycosides (e.g., neomycin, gentamicin,tobramycin); β-lactamase inhibitors (e.g., clavulanic acid, sulbactam);glycopeptides (e.g., vancomycin, teicoplanin, polymixin); ansamycins;bacitracin; carbacephem; carbapenems; cephalosporins (e.g., cefazolin,cefaclor, cefditoren, ceftobiprole, cefuroxime, cefotaxime, cefipeme,cefadroxil, cefoxitin, cefprozil, cefdinir); gramicidin; isoniazid;linezolid; macrolides (e.g., erythromycin, clarithromycin,azithromycin); mupirocin; penicillins (e.g., amoxicillin, ampicillin,cloxacillin, dicloxacillin, flucloxaciUin, oxacillin, piperacillin);oxolinic acid; polypeptides (e.g., bacitracin, polymyxin B); quinolones(e.g., ciprofloxacin, nalidixic acid, enoxacin, gatifloxacin, levaquin,ofloxacin, etc.); sulfonamides (e.g., sulfasalazine, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole), sulfadiazine);tetracyclines (e.g., doxycyline, minocycline, tetracycline, etc.);monobactams such as aztreonam; chloramphenicol; lincomycin; clindamycin;ethambutol; mupirocin; metronidazole; pefloxacin; pyrazinamide;thiamphenicol; rifampicin; thiamphenicl; dapsone; clofazimine;quinupristin; metronidazole; linezolid; isoniazid; piracil; novobiocin;trimethoprim; fosfomycin; fusidic acid; or other topical antibiotics.Optionally, the antibiotic agents may also be antimicrobial peptidessuch as defensins, magainin and nisin; or lytic bacteriophage. Theantibiotic agents can also be the combinations of any of the agentslisted above.

In one embodiment, the therapeutic agent is doxorubicin. Doxorubicin isan anthracycline antibiotic and anti-tumor agent that intercalates DNA.It is effective against cancers that cause solid tumor formation as wellas those that cause hematological malignancies (Booser & Hortobagyi,Drugs 47:223 (1994); Serpe et al., Eur. J. Pharm. Biopharm. 58:673(2004), which are hereby incorporated by reference in their entirety).Its administration in solution as doxorubicin.HCl (DOX.HCl) causes manyside effects, the most serious of which are cardiotoxicity, andmyelosuppression (Subedi et al., Eur. J. Pharm. Sci. 37:508 (2009); Zaraet al., Pharmacol. Res. 40:281 (1999), which are hereby incorporated byreference in their entirety). Improved safety can be achieved whendoxorubicin is administered in lanthanoid-DOTA derivatives complexencapsulating SLNs. Free base DOX can be used for entrapment in the SLNcore rather than its salts to avoid anions that could displace [LnDhS]⁻during preparation of the core-capsule SLN.

The solid lipid nanoparticles are biodegradable and can dissolve intocomponent small molecules that can be then cleared, likely through thekidneys. The lanthanoid-complex capsules are similarly biodegradable andcan be cleared through the kidneys as molecular degradation products.

The hyperbranched shell encapsulating solid lipid nanoparticle describedherein can be administered by various routes known to skilled in theart. One route is through intravenous administration. With normal kidneyfunction, the gadolinium complexes should be eliminated from thecirculatory system within 2-3 hours to avoid the accumulation of freeGd³⁺ ions, which can cause nephrogenic systemic fibrosis (NSF)(Bongartz, Magn. Reson. Mater. Phy. 20:57 (2007); Penfield & Reilly,Nat. Clin. Pract. Nephr. 3:654 (2007), which are hereby incorporated byreference in their entirety). Following injection, the SLNs, which arerelatively large, are confined to the circulatory system except wherethe leaky vasculature at solid tumor sites allows particles toextravasate, in some cases even as their diameters approach 600 nm (Yuanet al., Cancer Res. 55:3752 (1995), which is hereby incorporated byreference in its entirety). Particles that remain in circulation aretaken up by the mononuclear phagocyte system (MPS) (Wisse et al.,Toxicol. Pathol. 24:100 (1996), which is hereby incorporated byreference in its entirety). Rapid clearance of Gd³⁺ complexes typicallymeans that the GdDOTA capsule be readily hydrolyzed in the MPS. Thiscleavage can occur for ester links through acid hydrolysis in theendosome/lysosome compartments of leukocytes (Blasi et al., Adv. DrugDeliver. Rev. 59:454 (2007), which is hereby incorporated by referencein its entirety).

Intracellular biodegradation can occur either by disulfide bondreduction in the presence of glutathione in the cytosol or by esterhydrolysis in lysosome compartments of the cell (Lee et al.,Bioconjugate Chem. 18:13 (2007); Roos et al., Blood 53:851 (1979), whichare hereby incorporated by reference in their entirety). Hence cleavageof the capsule can occur followed by rapid clearance of the Ln-DOTAderivative complex via the kidneys and the liver (Wedeking & Tweedle,Nucl. Med. Biol. 15:395 (1988); Bui et al., Pub. Lib. Sci. One 5 (2010),which are hereby incorporated by reference in their entirety). Clearanceof the solid lipid fraction would normally take place via the liver.

In one embodiment, to achieve biodegradability, the homoserine hydroxylgroups of Monomer 1 (LnDhS complex) are converted to ester acrylates(Monomer 2) (Scheme 3). The capsules are then assembled byphotocrosslinking with appropriate initiator (Nguyen & West,Biomaterials 23:4307 (2002); Sawhney et al., Macromolecules 26:581(1993), which are hereby incorporated by reference in their entirety).

In one embodiment, to achieve biodegradability, acrylate group ofMonomer 2 (LnDhS complex) is reacted with thiolacetic acid (Bullock etal., J. Am. Chem. Soc. 76:1828 (1954), which is hereby incorporated byreference in its entirety) followed by base hydrolysis to acquire thecorresponding thiolates. The thiolates in the Stem layer of the mixedmicelle (solid lipid nanoparticle) can be converted to a disulfidecrosslinkers to assemble the capsule (Scheme 4).

In one embodiment, to achieve biodegradability, the homoserine (LnDhScomplex) is directly converted to homocysteine by halogenation (Br, orI) using standard methods followed by reaction with thioacetate, andacid hydrolysis. Oxidation of the thiol groups will then lead, asdescribed above, to the formation of disulfide crosslinking groups toassemble the capsule.

Other aspect of the invention relates to a method of using the compoundor molecular complex of formula (I) or (III), the hyperbranchedpolymeric nanoparticle or nanocapsule formed from formula (IV) and (V),or the mixed micelle described herein in catalysis reaction.

Yet another aspect of the invention relates to a method of using thecompound or molecular complex of formula (I) or (III), the hyperbranchedpolymeric nanoparticle or nanocapsule formed from formula (IV) and (V),or the mixed micelle described herein in materials synthesis.

As used in this aspect of the invention, the following terms, unlessotherwise indicated, shall be understood to have the following meanings.If not defined otherwise herein, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art to which this invention belongs. In the eventthat there is a plurality of definitions for a term herein, those inthis section prevail unless stated otherwise.

The term “alkyl” refers to an aliphatic hydrocarbon group which may belinear, branched, or cyclic hydrocarbon structures and combinationsthereof. Representative alkyl groups are those having 24 or fewer carbonatoms, for instance, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, i-pentyl, n-hexyl, and the like. Lower alkylrefers to alkyl groups having about 1 to about 6 carbon atoms in thechain. Branched alkyl means that one or more lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain.

The term “hydroxyalkyl” means an alkyl group is substituted with one ormore hydroxy substituents, wherein the alkyl group is as hereindescribed.

The term “mercaptoalkyl” or “thioalkyl” means an alkyl group issubstituted with one or more mecaptan (thiol) substituents, wherein thealkyl group is as herein described. The term “radical of mercaptoalkyl”means a mercaptoalkyl where S atom carries a radical. For instance,radical of mercaptoethanyl refers to —CH₂H₄—S., wherein the —S. canreadily bond to another radical to crosslink with a compound ormolecular complex of formula (I), (II), or (III).

The term “alkylthioalkyl” means a thioalkyl group is substituted withone or more alkyl substituents, wherein the alkyl group is as hereindescribed. Particularly, the thio group of the thioalkyl can besubstituted with one or more alkyl substituents.

The above “alkyl”, “hydroxyalkyl”, “mercaptoalkyl”, “radical ofmercaptoalkyl”, and “alkylthioalkyl” may be optionally substituted.

The term “arylalkyl” means an alkyl residue attached to an aryl ring.Examples are benzyl, phenethyl, and the like.

The term “substituted” or “optionally substituted” is used to indicatethat a group may have a substituent at each substitutable atom of thegroup (including more than one substituent on a single atom), providedthat the designated atom's normal valency is not exceeded and theidentity of each substituent is independent of the others. In accordancewith the present invention, up to three H atoms in each residue can bereplaced with alkyl, halogen, haloalkyl, alkyenyl, haloalkenyl,cycloalkyl, cycloalkenyl, hydroxy, alkoxy, acyl, carboxy, carboalkoxy(also referred to as alkoxycarbonyl), carboxamido (also referred to asalkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino,dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino,amidino, aryl, heteroaryl, aryloxy, or heteroaryloxy. When a substituentis keto (i.e., ═O), then two hydrogens on the atom are replaced.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds; by “stable compound” or“stable structure” is meant a compound that is sufficiently robust tosurvive isolation to a useful degree of purity from a reaction mixture,and formulation into an efficacious therapeutic agent.

In the characterization of some of the substituents, certainsubstituents may combine to form rings. Unless stated otherwise, it isintended that such rings may exhibit various degrees of unsaturation(from fully saturated to fully unsaturated), may include heteroatoms andmay be substituted with other substituent groups as described above.

The term “cation” refers to an ionic species (i.e., an atom or a groupof atoms) carrying a positive charge. Exemplary cations are H⁺, H₃O⁺,Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Ag⁺, Mg²⁺, Ca²⁺, Cu²⁺, R₃NH⁺, R₂NH₂ ⁺, RNH₃ ⁺(e.g., the protons of an alkylamine), etc.

The term “polymer precursor group” refers to a functional group for acompound or molecular complex (e.g., as shown in Formula (I) or (III))that is able to self-crosslink or crosslink with the neighboringcompound or molecular complex in presence of one or more crosslinkingagent to form a polymer. For instance, —SH or —S. can be used as apolymer precursor for a compound or molecular complex toself-crosslinking through formation of disulfide bonds; —OH can be usedas a polymer precursor for a compound or molecular complex in presenceof appropriate crosslinking agent to form a polymer through formation ofether, ester, thioester, amide, disulfide bonds or combinations thereof.

The present invention may be further illustrated by reference to thefollowing examples.

EXAMPLES

The following examples are intended to illustrate, but by no means areintended to limit, the scope of the present invention as set forth inthe appended claims.

Example 1 Materials

Emulsifying wax was obtained from Spectrum Chemicals. Polysorbatesurfactant (Brij® L23) and α-bromo-γ-butyrolactone were purchased fromSigma Aldrich Chemicals. Europium trichloride hexahydrate, gadoliniumtrichloride hexahydrate, divinyl sulfone (DVS) and potassium carbonatewere purchased from Alfa Aesar Chemicals. Hexadecyl trimethyl ammoniumchloride (CTA-Cl) was obtained from TCI America. All reagents were usedas received.

Example 2 Preparation of Cyclen Tetrabytyrolactone

Cyclen (1.00 g, 5.80 mmol), potassium carbonate (4.00 g, 29.02 mmol) andα-bromo-γ-butyrolactone (6.03 g, 36.57 mmol) were refluxed for 3 days inCH₃CN. The filtrate was collected and the solvent was removed by rotaryevaporation. A solid product was collected after severalether-wash/centrifugation cycles. The solid was redissolved in 15 mlchloroform, and extracted with 2×5 ml water. The chloroform solution wasdried with magnesium sulfate and the solvent was removed by rotaryevaporation to affort a yellow oil. Addition of diethyl ether producedthe product as a yellow powder in 71% yield.

¹H NMR (400 MHz, CDCl₃, d): 4.45-4.25 (m, 8H, O—CH₂), 3.95 (m, 4H,N—CH), 3.70-3.49 (m, 4H, cyclen-CH₂), 2.70-2.22 (m, 12H, cyclen-CH₂),2.40-2.25 (m, 8H, lactone-CH₂); ¹³C NMR (400 MHz, CDCl₃, d): 178.5(C═O), 65.18 (O—CH₂), 59.90 (N—CH), 47.22 (cyclen-CH₂), 21.25(lactone-CH₂). ESI-MS m/z: 508.9 [M+H]⁺, 531.9 [M+Na]⁺, 546.9 [M+K]⁺.

Example 3 Preparation of CTA-OH

CTA-Cl was stirred with KOH in anhydrous ethanol to prepare a stocksolution of ethanolic cetyltritnethylammonium hydroxide (CTA-OH). Thebyproduct, KCl(s), was removed by filtration. The sample was stored as a1 M stock solution in ethanol.

Example 4 Preparation of CTA[EuDOTA(N,N′,N′″,N″″-tetra-a-homoserine)](CTA[Eu(DhS)])

Cyclen tetrabutyrolactone (0.13 g, 0.26 mmol) and cetyltrimethylammoniumhydroxide (CTA-OH) (1.6 mmol) were combined in 10 ml of ethanol andstirred overnight to open the butyrolactone rings by base hydrolysis.The ethanol was removed by rotary evaporation. The product wasredissolved in 20 mL of methanol, and EuCl₃.6H₂O was added in 1:1.1ratio (0.105 mg, 0.29 mmol), under stirring for 3 hours. Methanol wasremoved by rotary evaporation. The remaining solid was redissolved in 25mL of ethanol. After refluxing for 1 hour, 0.19 g CTA[Eu(DhS)], a 74%yield, was obtained as precipitate, leaving CTACl byproduct in thesolution.

¹H NMR (400 MHz, D₂O, d): 41.9 (s, 4H, ring CH axial, minor isomer),22.0 (s, 4H, ring CH axial, major isomer). 8.1 (s, 1H, ring CH axialmajor isomer), 4.1 (s, 4H, α-CH (homoserine sidechain)), 3.48 (m, 2H,α-N—CH₂ (CTA)), 3.0 (s, 9H, CH₃ (N—CH₃ from CTA), 1.6 (s, 2H, (3-N—CH₂(CTA)), 1.2-1.0 (d, 26H, —CH₂— (CTA backbone)), 0.79 (m, 3H, —CH₃ (CTAterminal methyl group)), −0.5 (s, 8H, b-CH (homoserine sidechain)), −1.1(s, 4H, γ-CH (homoserine sidechain)), −2.0 (s, 4H, γ-CH (homoserinesidechain)), −3.1 (s, 4H, ring CH equatorial, major isomer), −4.2 (s,4H, ring CH axial, major isomer), −4.9 (s, 4H, ring CH equatorial, minorisomer), −8.6 (s, 4H, ring CH equatorial, major isomer), −12.1 (s, 4H,CH), −25.4 (s, 4H, CH-minor isomer). ESI-MS m/z: 729.0 [M]⁻.

Example 5 Preparation of CTA[GdDOTA(N,N′,N′″,N″″-tetra-a-homoserine)](CTA[Gd(DhS)])

The product CTA[Gd(DhS)] was obtained from GdCl₃.6H₂O (0.29 mmol) by themethod described above in Examples 2-4 in the synthesis of the europiumcomplex. ESI-MS m/z: 734.3 [M]⁻.

Example 6 Formation of [EuDhS]SLN using Brij® L23

The solid lipid nanoparticles were prepared in a microemulsion followingthe method of Oyewumi and Mumper (Oyewumi & Mumper, Drug Dev. Ind.Pharm. 28:317 (2002), which is hereby incorporated by reference in itsentirety). Solutions of (i) emulsifying wax in chloroform and (ii) otherreagents in water were freshly prepared before the synthesis.Emulsifying wax, 4 mg in 60 μl of chloroform from a stock solution, wasdeposited in a glass vial by evaporating the chloroform solvent. Thesolidified wax in the glass vial was heated to 55° C., and 1 mldistilled water was added dropwise while stirring at 1500 rpm andmaintaining the temperature at 55° C. A homogenous milky slurry wasformed, at which point 60 μL of 100 mM Brij® L23 (C₁₂H₂₅(OC₂H₄)₂₃OH)solution (6 μmol) was added. After 2 minutes, 1200 μL of 50 mMCTA⁺[Eu(DOTA)(N,N′,N′″,N″″-tetra-a-homoserine)]⁻ solution (60 μmol) wasadded. The solution was stirred until it became transparent (about 30minutes) and then was cooled on an ice bath to form SLNs. The SLNcolloidal solution was then diluted with the addition of 1 ml cold waterat 4° C. and filtered through a 0.22μ pore size membrane to remove largeparticles. The obtained SLN solution was stored at 4° C. for furtheruse.

Example 7 Formation of [EuDhS]_(CL)SLN

Crosslinking of the surface hydroxyls of [EuDhS]⁻ was carried out byadding divinylsulfone (DVS) to the solution of [EuDhS]SLN, such that DVSand CTA[Eu(DhS)] surfactant are in 1:4.17 molar ratio. The pH of the[EuDhS]SLN solution was raised to a value of 12.0 by adding 1.0 M NaOHsolution dropwise. After the desired pH was reached, 25 μL DVS solution(0.25 mmol) was added very slowly over a period of 5-10 minutes. Theresulting solution was allowed to react for 3-6 hours. After that, thepH was lowered to 7.5-8 by the slow addition of 1.0 M HCl (aq.). Sampleswere stored as aqueous colloidal solutions at 4° C. The samples remainedtransparent for at least 8 weeks.

Example 8 Characterization

Proton NMR data were obtained on a Bruker Avance 400 MHz NMRspectrometer. Relaxivity measurements were carried out in deionizedwater (H₂O) solutions at 25° C. with the temperature controlled by aBVT-3200. The aqueous (H₂O) samples were contained in the internalcapillary tubes of a coaxial cell with the D₂O lock solvent in theexterior (5 mm o.d.) chamber. Acquisition parameters were: time domain16K complex data point; 6410.26 Hz sweep width; 90° pulse with a lengthof 9 μs, at a power level of 5.00 dB repetition time T_(R) of 45 s;variable inversion time delay (τ) ranging from 100 ms to 5 s. Spectrawere processed with XWIN-NMR version 3.5.6 to obtain relaxation curves.

Electrospray ionization (ESI) mass spectrometric data were obtainedusing an Agilent 1100 Series Capillary LCMSD Trap XCT MS spectrometer.

Inductively coupled plasma optical emission spectroscopy (ICP-OES)chemical analyses were carried out by Galbraith Laboratories.

TEM images were acquired on a Philips CM-12 electron microscope. Sampleswere prepared on carbon coated copper grids and the micrographs wererecorded on a Gatan 1k×1k digital camera.

Fluorescence data was collected on a Hitachi F-2500 fluorescencespectrophotometer.

Dynamic light scattering measurements were carried out on a BrookhavenInstruments ZetaPALS/Zeta Potential Analyzer and a Beckman-Coulter N4Plus instrument.

Example 9 Ligand Synthesis and Particle Formation

Cationic SLNs have been employed primarily as vehicles for DNA and RNAtransfer (Kim et al., Mol. Pharm. 5:622 (2008); Tabatt et al., Eur. J.Pharm. Biopharm. 57:155 (2004); Siddiqui et al., Int. J. Pharm. 400:251(2010), which are hereby incorporated by reference in their entirety).In that capacity they provide matrices for the adsorption and deliveryof nucleic acid oligomers via endocytosis of the SLN. The desirablecationic surfactants for the fabrication of gene transfer SLNs arebiodegradable and non-cytotoxic as a result of their twin-tailed lipidstructure (Tabatt et al., Eur. J. Pharm. Biopharm. 57:155 (2004), whichis hereby incorporated by reference in its entirety). These surfactantsare paired with simple halide counterions that are later replaced bypolyanionic DNA. Satisfactory cationic SLNs for in vitro studies arealso available with simple single-tailed cationic surfactants such ascetyltrimethylammonium bromide (CTAB). The halides are later replaced bypolyanionic DNA. CTA⁺ was used in Examples 1-8, but with a uniqueapproach that substituted functional anions for simple halides such asBr⁻. The functional anions then provided precursor monomers forpolyanionic shell formation.

The work in Examples 1-8 focuses on the incorporation of EuDOTA inSLN-based particles. Early fluorescence measurements suggested that theuptake of anionic hydrophilic LnDOTA complexes into a hydrophobic SLNcore was unfavorable. The work described herein, however, demonstratesan unprecedented method, by which the LnDOTA complex nanocapsulessynthesized were assembled on a charged colloidal surface and thencrosslinked to form a hyperbranched enclosure around the SLN. Briefly,cationic surfactants were employed to stabilize the SLN surface(Eccleston, Colloid. Surface. A 123:169 (1997); Kim et al., Mol. Pharm.5:622 (2008); Tabatt et al., Eur. J. Pharm. Biopharm. 57:155 (2004);Siddiqui et al., Int. J. Pharm. 400:251 (2010), which are herebyincorporated by reference in their entirety). The counterions consistedof the monoanionic complex [EuDOTA(homoserine)-4⁻ ([EuDhS]⁻), in whichfour hydroxyethyl groups are attached to the α carbons of DOTA. Amicelle coronal crosslinking method was employed to form the capsule(Liu et al., Langmuir 18:7780 (2002); Read & Armes, Chem. Comm. 3021(2007), which are hereby incorporated by reference in their entirety).

This system is unique in its incorporation of[Eu(DOTA-N,N′,N″,N′″-tetrahomoserine)]-([EuDhS]⁻) as a monoanioniccounterion to CTA⁺ (Scheme 5).

It differs from preceding work in that it entails the assembly ofcomplex counterions in a polymeric network on a solid lipid nanoparticle(SLN) surface (Gao et al., J. Colloid Interf. Sci., 273:626 (2004),which is hereby incorporated by reference in its entirety). This systemappears to be the first capsule formed by crosslinking the lanthanideDOTA complexes; this system also appears to be the first capsuleformation around a solid lipid nanoparticle to generate amultimodal/multifunctional particle by virtue of the presence offunctional groups in the capsule layer.

[EuDhS]⁻, the complex serving as the functional anion, was prepared byreacting R,S-bromobutyro lactone with cyclen to formDOTA-N,N′,N″,N′″-tetrabutyrolactone, similar to the method described inLazlo et al. (Lazar et al., Eur. I Org. Chem. 351 (2002), which ishereby incorporated by reference in its entirety). Lactone ring-openingby base hydrolysis with CTA(OH) gave a cyclic octadentateDOTA-tetrahomoserine derivative. The europium compound EuCl₃ was thenadded to form the complex [EuDhS]⁻ in which europium was coordinated bythe four amine groups and four carboxylate groups to form aneight-coordinate EuDOTA complex (Scheme 2). In the formation and workupof the complex, three equivalents of the CTACl byproduct were removed,leaving one equivalent of CTA to form the isolable salt CTA⁺[EuDhS]⁻.The four pendant hydroxyethyl groups of the ligand were utilized to forma hyperbranched poly EuDOTA capsule.

The solid lipid nanoparticle core was prepared from emulsifying wax andBrij® L23, using a method similar as described in Oyewumi and Mumper(Oyewumi & Mumper, Drug Dev. Ind. Pharm. 28:317 (2002), which is herebyincorporated by reference in its entirety). Emulsifying wax (EW) is amulticomponent matrix that consists of cetylstearyl alcohol andpolysorbate 60 (USP28-NF23, 2005 Edition of the United StatesPharmacopeia and National Formulary (USP-NF), page 3107). The waxresists crystallization and therefore provides an amorphous matrix thatfavors the inclusion of additives (Ulrich, Biosci. Rep. 22:129 (2002),which is hereby incorporated by reference in its entirety). EWnanoparticles were formed in an oil/water (o/w) microemulsion at 55° C.The o/w emulsion was stabilized by the addition of the surfactants Brij®L23 and cetyltrimethylammonium (CTA⁺). Subsequent rapid cooling from 55°C. to 4° C. solidified the molten wax to yield cationic SLNs. Examples1-8 use CTA⁺[EuDhS]⁻/Brij® L23 mixed surfactant system for the SLN.Although CTA⁺Br⁻/Brij 35 mixed micelles were described in Gao et al.(Gao et al., J. Colloid Interf. Sci., 273:626 (2004), which is herebyincorporated by reference in its entirety), the surfactant systemprepared in Examples 1-8 uses a unique approach that employs afunctional anion instead of simple halides. This functional anion,[EuDhS]⁻, provided precursor monomers for polyanionic shell formation.

After the forming of the SLN colloidal solution of CTA⁺[EuDhS]⁻/Brij®L23, the crosslinking agent divinyl sulfone (DVS) was added to the SLNcolloidal solution to generate a hyperbranched polymer shell from themonoanionic europium complexes [EuDhS]⁻. The shell enclosed the cationicSLN to form an electrostatically associated core-shell assembly. SeeFIG. 4.

Several literatures have described MRI contrast-enhancing SLNs. Theseinclude the work of Morel et al. that reported the incorporation ofGdDTPA and GdDOTA in SLNs as MRI contrast agents (Morel et al., Eur. J.Pharm. Biopharm. 45:157 (1998), which is hereby incorporated byreference in its entirety). Report by Zhu et al. provided a detailedanalysis of an emulsifying wax SLN bearing a surfactant with a GdDTPAheadgroup (Zhu et al., J. Nanosci. Nanotechnol. 6:996 (2006); Sun etal., Magn. Reson. Med. 65:673 (2011), which are hereby incorporated byreference in their entirety). Another report described a similar studywith the improvement that the SLN is cationic that associated with thedianionic contrast agent [GdDTPA]² (Chen & Zhang, Chinese J. New Drugs18:1443 (2009), which is hereby incorporated by reference in itsentirety). For all these above systems described in the literature, thelocations and related relaxivity of Gd complexes have not beendiscussed.

In contrast, the spatial distribution of fluorescent lanthanide DOTAcomplex relative to the SLN core has been described herein. Theassociation of (CTA)[EuDhS] with an emulsifying wax SLN formed frommolten microemulsion is expected to yield a stable composition inelectrolyte-free aqueous solution. The Stern layer of the particle wouldconsist of surface CTA ammonium headgroups interspersed with the anioniceuropium complexes (Schemes 5 and 6).

In forming a polymerized capsule of [EuDhS]⁻ around an SLN core, Brij®L23 and CTA[EuDhS] were combined with molten emulsifying wax in anoil-in-water (o/w) microemulsion. Upon solidification, the resultingnanoparticles consisted of a core of emulsifying wax in which thelipophilic tails of CTA (—C₁₆H₃₃) were anchored and interspersed withthe lipophilic tails of Brij® L23 (—C₁₂H₂₅). The anionic counterion wascrosslinked to form mixed micelles that combined charged surfactants andcounterions with neutral, unreactive and bulky diblock copolymersurfactants such as Brij® L23. This SLN system was selected forbiomedical applications because of its core stability, functionality,and size selection provided by SLNs.

Charged surfactants can either form a micelle or stabilize the surfaceof an SLN core by using surfactants. However, the stable, functional SLNsystem described herein cannot be readily achievable by micelles formedthrough simpling mixing surfactants. Without crosslinking the anions,the [EuDhS]⁻ anions were expected to form mobile species in the Sternlayer with the CTA headgroup cations. The route to [EuDhS]⁻incorporation in an SLN-enclosing capsule includes a precursor in whichthe complex is partitioned between the Stern layer and the double layerin the vicinity of the SLN core. In this spatial arrangement, the anionswithin the Stem layer are, by design, sterically isolated by the Brij®L23 polyether-(C₂H₄O)₂₃—OH chains. In aqueous solution at high ionicstrength, anion exchange is expected to displace much of the complexfrom the particle surface. The assembly is also unstable in simpleaqueous media on a longer time scale because of the disintegration oraggregation of the SLN cores (Oyewumi & Mumper, Drug Dev. Ind. Pharm.28:317 (2002), which is hereby incorporated by reference in itsentirety). As a remedy, the SLN assembly can be stabilized against bothanion diffusion and core disintegration or aggregation by crosslinkingthe anionic complexes. This process can be described as emulsion surfacepolymerization (ESP).

The crosslinking reaction was carried out in aqueous solution by thereaction of divinyl sulfone (DVS) with the pendant hydroxyethyl groupsof the anionic [EuDhS]⁻ complexes. Through this approach a capsule wasformed by a hyperbranched polymer network enclosing a cationic SLN,[EuDhS]_(CL)SLN (Scheme 6). Although a crosslinking reaction in blockcopolymer micelles has been reported (Liu et al., Langmuir 18:7780(2002); Read & Armes, Chem. Comm. 3021 (2007), which are herebyincorporated by reference in their entirety), such reactions have notbeen used to crosslink SLN to stabilize the SLN assembly.

In the solid EW particle the lipophilic tails of CTA⁺ (—C₁₆H₃₃) and BrijL23 (—C₁₂H₂₅) are embedded in the lipid, leaving the cationic headgroupof CTA⁺ and the polyoxomer block of Brij L23 exposed to the aqueous bulkphase. The [EuDhS]⁻ counteranion monomers are expected to occupy theStem layer as mobile species that exchange among fixed-position cationicCTA headgroups of the particle (CTA[EuDhS]SLN). The anionic complexes,[EuDhS]⁻ are subject to mass action involving small ions, such as Cl⁻ inhigh salt solutions, that can displace [EuDhS]⁻ complexes from the SLNsurface. As with SLNs in general, the NP, CTA[EuDhS]SLN, is unstable insimple aqueous media on longer time scales because of the well knowntendency for disintegration or aggregation. Crosslinking to form thehybrid particle (CTA)[EuDhS]_(CL)SLN prevented anion displacement, andstabilized the particles against core disintegration and aggregation.

The crosslinking step was carried out at room temperature afterformation of solid lipid nanoparticles. Crosslinking produces ahyperbranched polymer network that encloses the cationic SLN to form thehybrid NP [EuDhS]_(CL)SLN (Scheme 6). The anionic complex monomerswithin the Stern layers of adjacent particles are sterically separatedby the polyether —(C₂H₄O)₂₃—OH chains of Brij L23, which preventsinter-particle crosslinking.

Example 10 Europium Fluorescence

In order to validate the requirement for crosslinking to stabilize theassembly, fluorescence was used to monitor the displacement of europiumcomplexes from the SLN surface in an aqueous salt solution, comparingthe SLN system with and without crosslinking. Excitation of the [EuDhS]⁻complex at 319 nm generated emission peaks at 578, 592 and 617 nm. Thefluorescent emission of the core solid lipid at 406 nm did not interferewith measurements in the region of the europium emission. The [EuDhS]SLNsample was dialyzed against approximately 50 ml 0.68 M sodium chloride,a 2000 fold excess of Na⁺, and compared to a salt-free [EuDhS]SLNcontrol that was dialyzed against distilled water, using a 2.0 kDacutoff dialysis membrane. After 24 hours of dialysis, the europiumemission peak at 617 nm decreased by 94%; the fluorescence of thecontrol was unchanged (FIG. 5). The loss of fluorescence intensityindicates the displacement of [EuDhS]⁻ complex from the [EuDhS]SLNsurface by aqueous Cl⁻, when there is no crosslinking.

Emulsion surface polymerization (ESP) was conducted to the SLN system byadding divinyl sulfone (DVS) to a solution of [EuDhS]SLN. Thecross-linking step was allowed to proceed at room temperature for aperiod of 3-6 hours. The crosslinked product, [EuDhS]_(CL)SLN, waspurified by dialysis against distilled water, and stored as an aqueoussolution under refrigeration. It was then tested for stability bydialysis against aqueous NaCl using the procedures as described above.As shown in FIG. 6, in the crosslinked sample, [EuDhS]_(CL)SLN, only 19%of the [EuDhS]⁻ complexes were displaced by Cl⁻ (FIG. 6). These resultsshow that the Eu complexes reside only within the Stern and double layerregions at the SLN surface. The results prove the efficacy of [EuDhS]⁻crosslinking in forming a stable capsule that encloses the cationic SLNcore. Further, based on transmission electron microscopic (TEM) data(FIG. 7), crosslinking between adjacent SLN particles is absent, likelythe outcome of Brij® L23 steric barriers.

Further, the concentration of Eu complexes in the solid lipid core mustbe negligible because Eu fluorescence is absent after the displacementof [EuDhS]⁻ by Cl⁻ ions. Support for this conclusion is provided by areport that europium complexes exhibit fluorescence of comparableintensity both in solution and in sol-gel derived materials (Matthews etal., Chem. Mater. 5(12): 1697-1700 (1993), which is hereby incorporatedby reference in its entirety.). Therefore it was believed thatsignificant amounts of [EuDhS]⁻ within the SLN matrix would beobservable by fluorescence. This fluorescence would persist even afterthe displacement by Cl⁻ of [EuDhS]⁻ complexes in the Stern layerexternal to the SLN core.

Example 11 Solid Lipid Nanoparticle Characteristics

An average hydrodynamic diameter of 51.0±10.9 nm (polydispersity indexof 0.6) was found for [EuDhS]_(CL)SLN by dynamic light scattering (DLS)measurements. A zeta potential of +10.7 mV was measured by Doppler shiftlight scattering at pH 7.0. TEM images show that particles arepolydisperse, but most abundant in the 50 nm range and with nearlyspherical geometry.

The number of SLN particles per sample was determined from the totalmass of emulsifying wax in the sample, the density of the wax, 0.85g/cm³, and the average particle size as determined by DLS and TEM data.The result shows there are 1.64×10¹³ particles per mL aqueous solution.The concentration of [EuDhS]⁻ was determined from magneticsusceptibility measurements to be 0.73 mM 2.64×10¹⁷ ions in a samplecontaining 1.64×10¹³ particles, where the effective magnetic moment ofEu³⁺ is 3.40 at 25° C. (Peters et al., Prog. Nucl. Magn. Reson.Spectrosc. 28:283 (1996), which is hereby incorporated by reference inits entirety). The average number of [EuDhS]⁻ ions per [EuDhS]_(CL)SLNshell is therefore 8×10³ ions for a 65 nm diameter particle. This issmaller than the 34×10³ ions expected (Siddiqui et al., J. ColloidInterf. Sci. 337:88 (2009); Zhu et al., J. Nanosci. Nanotechnol. 6:996(2006), which are hereby incorporated by reference in their entirety).Perhaps the degree of hyperbranched crosslinking of the SLN assembly isless than ideal due to a combination of factors of steric barriers, DVShydrolysis or suboptimal reactant ratios.

Additional dynamic light scattering measurements also gave an averagehydrodynamic diameter of 65.1±7.3 nm for [EuDhS]_(CL)SLN. The zetapotential of the crosslinked particle was +10.7 mV as measured byDoppler shift light scattering at pH 7.0. The particles werepolydisperse by transmission electron microscopy, but were most abundantin the 50 nm range and with nearly spherical geometry. Smaller sphericalparticles were observed with diameters in the 5-10 nm range while largerparticles appeared to be aggregates with diameters in the range of 100nm. See FIG. 7.

From ICP-OES analysis of 5 mg of [EuDhS]_(CL)SLN, europium was found atthe level of 187 ppm (1.23 μmol) which corresponds to 7.41×10¹⁷ Eu ionsin the sample. Assuming a uniform particle density of 0.85 g/cm³, and anaverage diameter of 65.1 nm, the 5 mg sample contains 4.07×10¹³ SLNparticles with an average of 1.8×10⁴ Eu complexes per particle.

Example 12 NMR Analysis

Europium (III) is paramagnetic due to low-lying states with J>0, justabove the diamagnetic ⁷F₀ ground state. In [EuDhS]⁻, the Eu³⁺ ion servesas a shift reagent, which complements its fluorescent properties. Freecomplexes exhibit a room temperature ¹NMR spectrum that is broadlycongruent with that reported earlier for [EuDOTA]⁻ (Aime et al., Inorg.Chem. 36:2059 (1997); Desreux, Inorg. Chem. 19:1319 (1980); Aime et al.,Inorg. Chem. 31:4291 (1992); Woods et al., J. Am. Chem. Soc. 122:9781(2000), which are hereby incorporated by reference in their entirety).The expected dipolar shifts of cyclen protons induced by europiumappeared as a range of peaks in the upfield 0-(−11) ppm region, and apeak with a shoulder 21 ppm downfield with a peak from the adventitiousproton peak at 4.7 ppm in D₂O solvent (FIG. 8). As shown in FIG. 8, thepeaks of the shell-localized complexes of [EuDhS]_(CL)SLN with largeupfield and downfield displacements were congruent with thecorresponding peaks of the free complex [EuDhS]⁻ (inset); peaks in the0-7 ppm downfield region are assigned to homoserine sidechain,crosslinkers and SLN core protons, as would be expected with thedistance attenuation of dipolar shifts. Peak assignments cannot beobtained from room temperature spectra of racemic EuDOTA derivativesbecause of structural isomerization, in which case peaks from thediasteriomers δδδδ, δλλλ, δδλλ and δλδλ overlap (Woods et al., J. Am.Chem. Soc. 122:9781 (2000), which is hereby incorporated by reference inits entirety).

Example 13 Gadolinium Relaxivity

Liposomal MRI contrast agents have been described to incorporate Gdcomplexes at both the inner and outer lipid/water interfaces. Asexpected, the relaxivity for the inner interface diminishes by thedecreased rate of water exchange between the inner and outer aqueousregions (Muller et al., Langmuir 24:4347 (2008), which is herebyincorporated by reference in its entirety). An earlier study describedgadolinium complexes in SLNs (Morel et al., Eur. I Pharm. Biopharm.45:157 (1998), which is hereby incorporated by reference in itsentirety). However, whether it can be used as contrast agent and themechanism of contrast enhancement are unknown, because the locations ofthe polar Gd contrast agents were unknown in that study. Zhu et al.employed a lipid tethered GdDTPA complex that was incorporated in thesurfactant layer of an SLN (Zhu et al., J. Nanosci. Nanotechnol. 6:996(2006), which is hereby incorporated by reference in its entirety).However, the convenience and efficacy of this approach may be greatlyoffset by the expense of lipid precursors. It is therefore desirable toattach Gd complexes by simple, but easily accessible linkages.

As shown by Morel et al., gadolinium complexes associated with solidlipid nanoparticles generally display T₁ relaxivities comparable tosimple molecular agents in aqueous solution (Morel et al., Eur. I Pharm.Biopharm. 45:157 (1998), which is hereby incorporated by reference inits entirety). This result suggests that active Gd complexes reside atthe lipid/water interface. Interior Gd complexes will not likely beaccessible for water exchange, and thus will not induce inner sphererelaxation of water proton spins. Morel et al. offered two alternativeexplanations for the mono-exponential free induction decay of the waterproton magnetization in their Gd/SLN system. One suggestion was that theGd complexes were embedded in the lipid core with fast exchange of watermolecules occurring between the compartments. A second hypothesis wasthat no exchange takes place between the compartments; rather, only Gdcomplexes on the outer surface contributed to water relaxation. Bothexplanations reasonably imply that Gd³⁺ ions must be in proximity tobulk water to induce significant proton relaxation. To optimizerelaxivity for liposomal delivery vehicles, Gd complexes typically needto be attached to the outer layer of the liposomal bilayer liposomaldelivery vehicles (Kamaly & Miller, Int. J. Mol. Sci. 11:1759 (2010),which is hereby incorporated by reference in its entirety).

The congeners [GdDhS]⁻ and [GdDhS]_(CL)SLN were prepared from theproduct of GdCl₃.6H₂O and DhS⁴⁻. The r₁ relaxivities of the Gd[DhS]⁻ andGd[DhS]_(CL)SLN were found to be 4.16 and 7.70 s⁻¹ mM⁻¹, respectively,at 9.4 T (400 MHz) NMR and 25° C. (Table 1). The corresponding wholeparticle relaxivity of Gd[DhS]_(CL)SLN is r₁=2.09×10⁵ mM⁻¹.

TABLE 1 Relaxivity (r_(l)) per Gd, or per particle in H₂O at 9.4 T (400MHz), 25° C. Compound Complexes per particle Relaxivity (s⁻¹mM⁻¹)Gd[DTPA]²⁻ 4.6^(a) Gd[DhS]⁻ 4.16 Gd[DhS]_(CL)SLN 7.70 Gd[DhS]_(CL)SLN18000 209000^(b) (per particle) ^(a)Siddiqui et al., J. ColloidInterface Sci., 337(1): 88-96 (2009). ^(b)Relaxivity per particle isestimated from the Gd³⁺ population on the particle surface.

Several reports have described the use of SLNs for MRI contrastenhancement (Morel et al., Eur. J. Pharm. Biopharm. 45:157 (1998); Zhuet al, J. Nanosci. Nanotechnol. 6(4):996-1003 (2006); Sun et al., Magn.Reson. Med. 65(3):673-79 (2011); Chen et al., Zhongguo Xinyao Zazhi18(15):1443-47 (2009), all of which are hereby incorporated by referencein their entirety). In general for these systems the locations and/orrelaxivities of the Gd complexes were not directly discussed. Bycontrast, the work described here focused on the spatial distribution of[EuDhS]⁻ complex relative to the SLN core, by measuring the relatedrelaxivity of the congener [GdDhS]⁻ complex of the [LnDhS]_(CL)SLNsystem.

In the early work by Morel et al., gadolinium complexes associated withsolid lipid NPs had r₁ relaxivities comparable to simple molecularagents. Results from more recent studies suggest that r₁ relaxivityvalues ≧4-5 s⁻¹ mM⁻¹ derive only from surface localized Gd complexes.

In the system described in Examples 1-13, a method has been describedthat can significantly expand the role of SLNs by making them multimodalusing a simple and cost effective synthetic tools. In this method,cationic solid lipid nanoparticles have been prepared herein witheuropium DOTA complexes as counterions. The europium DOTA complexes havependant hydroxyl groups that act as precursors for Stern layerpolymerization using DVS crosslinking. The process is referred to asemulsion surface polymerization (ESP). Further, from the fluorescenceresults, the locations of fluorescent lanthanoid complexes relative toSLN have been readily ascertained. Additionally, this system provides aconvenient route to assemble a contrast agent with LnDOTA restricted tothe surface with the core reserved as a compartment for other componentssuch as drugs or secondary imaging agents. Relaxivity measurements showthe relaxivity of [Gd(DhS)]⁻ in the capsule, [Gd(DhS)]_(CL)SLN, to be7.70 s⁻¹mM⁻¹, which is significantly higher than that of the parent[Gd(DhS)]⁻ complex, 4.16 s⁻¹ mM⁻¹. This is a particularly encouragingresult for using this novel SLN system as new nanoscaleimaging/therapeutic agents for MRI, fluorescence or drug delivery.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A compound of formula (I) or (II):

wherein R is H or a cation; R′ is H, or a polymer precursor group whichis able to self-crosslink or crosslink in presence of one or morecrosslinking agents. 2-6. (canceled)
 7. A molecular complex comprising amolecule of formula (III):

and optionally a balancing counterion, wherein M is a chelating metalion and R′ is H, or a polymer precursor group that is able toself-crosslink or crosslink in presence of one or more crosslinkingagents.
 8. The molecular complex of claim 7, wherein the metal ion is alanthanoid.
 9. The molecular complex of claim 7, wherein the metal ionis La, Eu, Tb, Gd, Ga, or In.
 10. The molecular complex of claim 7,wherein R′ is H or substituted alkyl group.
 11. The molecular complex ofclaim 7, wherein R′ is hydroxyalkyl, mercaptoalkyl or radical ofmercaptoalkyl, or alkylthioalkyl.
 12. The molecular complex of claim 11,wherein R′ is —CH₂—OH, —C₂H₄—OH, —C₂H₄—S—CH₃, —C₂H₄—SH, or —C₂H₄—S. 13.(canceled)
 14. A hyperbranched polymeric nanoparticle or nanocapsuleformed from the molecular complex of claim
 7. 15. A method of making ahyperbranched polymeric nanoparticle or nanocapsule comprising:connecting the molecular complex of claim 7 by modifying the functionalgroup of R′ through ether, ester or disulfide bond formations. 16-18.(canceled)
 19. A hyperbranched polymer nanoparticle or nanocapsulecomprising: hyperbranched polymeric unit having formula (IV):

 and optionally a crosslinking moiety CL, wherein: R is H or a cation; Xis O or S; n is 1-10; p is the number of the units of formula (IV)ranging from 2 to 200,000; and crosslinking moiety CL, if present, is amoiety connecting the hyperbranched polymeric units together throughcovalently bonding to each X of formula (IV).
 20. A hyperbranchedpolymer nanoparticle or nanocapsule comprising: hyperbranched polymericunits having formula (V):

 and optionally a crosslinking moiety CL, wherein: M is a chelatingmetal ion; X is O or S; n is 1-10; p is the number of the units offormula (V) ranging from 2 to 200,000; and crosslinking moiety CL, ifpresent, is a moiety connecting the hyperbranched polymeric unitstogether through covalently bonding to each X of formula (V).
 21. Thehyperbranched polymeric nanoparticle or nanocapsule of claim 20, whereinX is S.
 22. The hyperbranched polymeric nanoparticle or nanocapsule ofclaim 20, wherein X is O, and the crosslinking agent is divinyl sulfone(DVS), dicarboxylic acid, diepoxybutane, diepoxyoctane, epichlorohydrin,butanediol-diglycidyl ether (BDDE), ethylene glycol diglycidyl ether,polyglycerol polyglycidyl ether, ethylene sulfide, glutaraldehyde,bromoacetic anhydride, acrylic anhydride, 3-mercaptopropanoate,thioacetic acid, or combinations thereof.
 23. A hyperbranched polymershell comprising the crosslinked hyperbranched polymeric nanoparticlesor nanocapsules of claim
 20. 24. A mixed micelle comprising anemulsifying wax as the micelle core; and at least one cationicsurfactant associated to the surface of the micelle core, wherein theanionic portion of the cationic surfactant is the hyperbranchedpolymeric nanoparticle of claim
 20. 25-27. (canceled)
 28. A solid lipidnanoparticle, comprising a therapeutic agent and the mixed micelle ofclaim
 24. 29-32. (canceled)
 33. A contrast agent comprising the mixedmicelle of claim
 24. 34. A method of preparing a solid lipidnanoparticle, comprising: formulating a micelle core by mixing aemulsifying wax, at least one neutral surfactant, and at least onecationic surfactant, wherein the anionic portion of the cationicsurfactant is the molecular complex of claim 7; crosslinking themolecular complexes by modifying the functional group of R′, optionallybonded through one or more crosslinking agents, to form a hyperbranchedpolymeric shell around the micelle core, thereby forming a solid lipidnanoparticle. 35-41. (canceled)
 42. A method of biomedical imaging,comprising the step of (a) administering to a patient the mixed micelleof claim 24, and (b) conducting a biomedical imaging test on thepatient. 43-44. (canceled)
 45. A drug delivery vehicle comprising thesolid lipid nanoparticle of claim
 28. 46. A method of delivering drugsacross the blood brain barrier, comprising the step of administering toa patient the solid lipid nanoparticle of claim
 28. 47. (canceled)
 48. Amethod of delivering a high concentration of contrast enhancing and/orimaging agents comprising the step of administering to a patient themixed micelle of claim 24, wherein the chelated metal acts as thecontrast enhancing and/or imaging agent.